I transferred my sample on a 0.2µm NC membrane (semidry blot), incubated with antibody (as usual) and did ECL but I saw some weird, white signals instead of black bands. I was able to detect the same samples on a gel stained with Coomassie, so there must be enough protein on the gel. So why do I have these weird signals after ECL (see attached picture)? Never had that before.
Can anyone suggest something, please.
I have also seen this. My interpretation is that there is a high background relative to the signal, and the proteins in the lane (the ones not detected by the antibody) act as blockers that reduce the background.
I have also seen this. My interpretation is that there is a high background relative to the signal, and the proteins in the lane (the ones not detected by the antibody) act as blockers that reduce the background.
Hi Adam; so you mean, there is maybe too much protein transferred to the membrane that block the binding of my antibody?
I would suggest using a mild detergent in the washing buffers instead of that you used which washed out your protein and bound antibody together
I don't think the problem is with too much protein. I think the problem is that the detection of the antigen is too weak.
It looks like the lanes with proteins is the best blocked portion of the membrane. Did you remember to block for 1 hour after transfer? Did you activate the membrane prior to transfer with alcohol? Did you have a mild detergent (tween) in with the primary and secondary antibody (should not with BSA block)?
Sorry its more Q than A! Good luck!
Fergal O'Farrell : Thanks for suggestions. Yes I did block the membrane after transfer before incubation with Ab with 5% milk (as usual). I did NOT activate the membrane ´cos it is a Nitrocellulose membrane and NOT a PVDF membrane. NC you don´t have to activate but to equilibrate a bit in transfer buffer (same as gel). I did not use Tween or Triton in the buffer this time. But today, I had some TX100 in it, and I got quite the same results. So, I think, it´s something else.
There are several reasons for this.
1. There is something wrong with your sample. I think you are loading too much proteins for the white lane. Gel 1, Probably around 100-250 ug is loaded in the white lane, That is too much for WB detection. Regarding the Gel 3, the sample is not prepared well for the white lane. You could see most of the protein is in the upper part of the gel when you coommassie staining and there are no clear bands below.
2. Check the antibody is good or not. You could use some positive cell lysates, recombinant proteins or ask the antibody company to help you figure this out.
Another interesting thing is your protein ladder is also white in the gel 1. I think it is maybe due to too strong signal.
Additionally, make sure all the reagents for WB are prepared well.
Good luck.
I have encountered it once due to the problem with the higher concentration of primary antibody. Try to use the new aliquot and use more diluted sample.
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